Chemistry
Chromatography
Chromatography is a technique used to separate and analyze mixtures of chemical substances. It works by passing a sample through a stationary phase, which causes the components to move at different rates, leading to their separation. This method is widely used in chemistry for identifying and quantifying the components of complex mixtures.
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11 Key excerpts on "Chromatography"
- Chavan, U D(Authors)
- 2021(Publication Date)
- Daya Publishing House(Publisher)
CHAPTER 8 Chromatography 1. Chromatography Principles and Applications Chromatography is the collective term for a set of laboratory techniques for the separation of mixtures . The mixture is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material called the stationary phase . The various constituents of the mixture travel at different speeds, causing them to separate. The separation is based on differential partitioning between the mobile and stationary phases. Subtle differences in a compound’s partition coefficient result in differential retention on the stationary phase and thus changing the separation. Chromatography may be preparative or analytical. The purpose of preparative Chromatography is to separate the components of a mixture for more advanced use (and is thus a form of purification ). Analytical Chromatography is done normally with smaller amounts of material and is for measuring the relative proportions of analytes in a mixture. The two are not mutually exclusive. History Thin layer Chromatography is used to separate components of a plant extract, illustrating the experiment with plant pigments that gave Chromatography its name. Chromatography was first employed in Russia by the Italian-born scientist Mikhail Tsvet in 1900. He continued to work with Chromatography in the first decade of the 20th century, primarily for the separation of plant pigments such as chlorophyll, carotenes, and xanthophylls . Since these components have different colours (green, orange, and yellow, respectively) they gave the technique its name. New types of Chromatography developed during the 1930s and 1940s made the technique useful for many separation processes. Chromatography technique developed substantially as a result of the work of Archer John Porter Martin and Richard Laurence Millington Synge during the 1940s and 1950s.
eBook - PDF- Raja Ghosh(Author)
- 2006(Publication Date)
- WSPC(Publisher)
151 Chapter 9 Chromatography 9.1. Introduction Chromatography is a solute fractionation technique which relies on the dynamic distribution of molecules to be separated between two phases: a stationary (or binding) phase and a mobile (or carrier) phase. In its simplest form, the stationary phase is particulate in nature. The particles are packed within a column in the form of a packed bed. The mobile phase is passed through the column, typically at a fixed velocity. A pulse of sample containing the molecules to be separated is injected into the column along with the mobile phase. The velocities at which these molecules move through the column depend on their respective interactions with the stationary phase. For instance, if a molecule does not interact with the stationary phase its velocity is almost the same as that of the mobile phase. With molecules that do interact with the stationary phase, the greater the extent of interaction, the slower is the velocity. This mode of chromatographic separation is also called pulse Chromatography to distinguish it from step Chromatography which is operated differently. Chromatography is used for the separation of different substances: proteins, nucleic acids, lipids, antibiotics, hormones, sugars, etc. When used for analysis of complex mixtures, Chromatography is referred to as analytical Chromatography while when used to separate molecules as part of a manufacturing process, it is referred to as preparative Chromatography. Some of the applications of Chromatography in biotechnology are listed below. 1. Biopharmaceutical production 2. Biopharmaceutical and biomedical analysis Principles of Bioseparations Engineering 152 3. Environmental analysis 4. Foods and nutraceuticals production 5. Diagnostics 6. Process monitoring 9.2. Chromatography system A chromatographic separation system consists of a column, mobile phase reservoir/s, pump/s, sample injector, detector/s and sometimes a fraction collector.
eBook - PDF- J.R.J. Paré, J.M.R. Bélanger(Authors)
- 1997(Publication Date)
- Elsevier Science(Publisher)
This naturally led to its counterpart: liquid Chromatography or high- performance Liquid Chromatography (HPLC); preferred technique for separation of non-volatile or thermally unstable species. This chapter will discuss planar Chromatography and column Chromatography in general. The more advanced techniques such as gas Chromatography (GC) and high performance liquid Chromatography (HPLC) will be discussed extensively in subsequent chapters. 1.2 Chromatography: A SEPARATION TECHNIQUE As mentioned previously, the objective of Chromatography is to separate the various substances that make up a mixture. The applications range from a simple verification of the purity of a given compound to the quantitative determination of the components of a mixture. The chromatographic system consists of a fixed phase (stationary phase) and a moving phase (mobile phase). The mixture to be analysed or solute is introduced into the system via the mobile phase, and it is the affinity of the solute for one phase over the other which will govern its separation from the other components. Each component is retained to a different degree in the system and retention is based on various attraction forces. This gives rise to different modes of separation which will be presented in this chapter. B~langer, Bissonnette, and Par~ 1.3 THEORY Chromatography is the name given to methods by which two or more components of a mixture are physically separated. The Chromatography system consist of a stationary phase which can be a solid or a liquid supported on a solid; and a mobile phase which flows continuously around the stationary phase. The mobile phase can be gaseous or liquid. Depending on the nature of the mobile and stationary phases, the following types of Chromatography can be achieved: Gas-Solid (GS), Gas-Liquid (GL), Liquid- Liquid (LL) and Liquid-Solid (LS) Chromatography.
- Prakash Singh Bisen, Anjana Sharma(Authors)
- 2012(Publication Date)
- CRC Press(Publisher)
Today, the technique has developed in all dimensions as perhaps the single most powerful analytical and preparative method available in the laboratory. 4.2 GENERAL PRINCIPLES The basis of all forms of Chromatography is the differential partition of a compound between two immiscible phases, one of which is stationary and the other, mobile. The way in which a compound is partitioned or distributed between two immiscible phases is given by the partition coefficient ( K d ): K d Concentration in phase A Concentration in phase B = The value of K d is constant at a given temperature. Depending on the different types of phases involved, there are different forms of Chromatography (Table 4.1). Depending on the mode by which separation is achieved, there are three types of Chromatography: 1. Column Chromatography, in which the stationary phase is packed into glass or metal col-umns and the mobile phase percolates through the column. 2. PC, in which the stationary phase is supported by cellulose fibers of paper and the mobile phase moves through the interstitial spaces by capillary action. Sometimes, the cellulose fibers of the paper act as the solid stationary phase. 3. Thin-layer Chromatography (TLC), in which the stationary phase is thinly coated onto glass plates and the mobile phase moves along the stationary phase. 62 Introduction to Instrumentation in Life Sciences 4.3 COLUMN Chromatography In the column Chromatography technique, the components of a mixture are separated in a column as distinct zones and each zone is eventually displaced from the column as a series of fractions (Figure 4.1). The apparatus and general techniques used for column adsorption partition, ion exchange, exclusion, and affinity Chromatography have much in common and are discussed in Section 4.3.1. Gas–liquid Chromatography (GLC) and high-performance liquid Chromatography (HPLC) have their own unique apparatus and procedures and are discussed separately.
eBook - PDF- Satinder Ahuja(Author)
- 2003(Publication Date)
- Academic Press(Publisher)
1 RELATING Chromatography TO SEPARATIONS I. DEFINING SEPARATION II. EVOLUTION OF Chromatography A. Definition of Chromatography B. Similarity of Chromatography to Separation Methods III. SEPARATIONS IN EVERYDAY LIFE IV. BASIS OF SEPARATIONS A. Physicochemical Phenomena B. Utilizing a Desirable Physical Property V. MODES OF Chromatography A. Adsorption Versus Absorption B. Partition or Distribution C. Exclusion D. Ion Exchange VI. UNIFIED SEPARATION SCIENCE VII. SELECTIVITYAND DETECTABILITY A. Selectivity B. Detectability REFERENCES QUESTIONS FOR REVIEW A chromatographic method can be considered simply a physical method of separation in which components to be separated are distributed between two phases. However, it should be recognized that chromatographic methods have evolved into complex and elegant methods that utilize a variety of physicochemical approaches to provide the desired separation of complex molecules (see Chapters 6–10). Chromatography can be related directly to some of the simple nonchromatographic separation methods by the same common basic physicochemical principles employed in these methodologies to achieve the desired results. We will see from the discussion in Sections I and II that separation is a broad term that can include nonchromatographic methods as well as all of the chromatographic methods, and it should be recognized at the very outset that this does not imply that all separation methods are chromatographic. Chromatography provides a variety of powerful methods that can help separate a large number of different compounds. As a matter of fact, it can be 1 said without much hesitation that a chromatographic method is likely to be the method of choice for complex samples when a selection has to be made among various separation methods. The contributions of Chromatography to various scientific disciplines are numerous. The investigations conducted with the chromatographic methods have resulted in a great variety of benefits to human beings.
eBook - PDF- Haleem J. Issaq(Author)
- 2001(Publication Date)
- CRC Press(Publisher)
1 Chromatography: The Separation Technique of the Twentieth Century* Leslie S. Ettre Yale University, New Haven, Connecticut An essential condition for all fruitful research is to have at one's disposal a satisfactory tech-nique. Tout progrès scientific est un progrès de méthode f as somebody once remarked. Unfortunately the methodology is frequently the weakest aspect of scientific investigations. M. S. Tswett [1] I. INTRODUCTION M. S. Tswett contemplated the possibility of Chromatography in 1899-1901 while carrying out his first research on the physico-chemical structure of plant chlorophylls, and he reported on a new category of adsorption analysis in 1903. Thus Chromatography was born with the twentieth century. Chromatography is based on a flow system containing two phases, mobile and stationary, and the sample components are separated according to differences in their distribution between the two phases. This separation principle is flexible and versatile, permitting one to explore various ways to achieve separation, and indeed, during its 100-year evolution Chromatography underwent many steps, each representing new approaches and further broadening the scope and application of the technique. Each step in this evolution followed logically from the previous one. It started as liquid adsorption Chromatography, which was followed by partition chromatog-raphy. Next its use was extended to the analysis of gases and vaporized samples. The principles * Originally published in Chromatographic!, 51:7-17, January 2000. Reprinted with permission from Vieweg Publishers, Wiesbaden, Germany. t All scientific progress is progress in a method. This statement is attributed to the French philosopher René Descartes (1596-1650), the author among others of the book Discours de la méthode. 1 2 ETTRE of separation were also broadened by adding ion exchange and separation by molecular size and, most recently, electroChromatography.
eBook - PDFBasic Bioscience Laboratory Techniques
A Pocket Guide
- Philip L.R. Bonner, Alan J. Hargreaves(Authors)
- 2022(Publication Date)
- Wiley-Blackwell(Publisher)
Basic Bioscience Laboratory Techniques: A Pocket Guide, Second Edition. Philip L.R. Bonner and Alan J. Hargreaves. © 2022 John Wiley & Sons Ltd. Published 2022 by John Wiley & Sons Ltd. 7 117 7.1 Introduction Chromatography was first developed by the Russian botanist Mikhail Tswett in the early 1900s when he produced a colour abundant separation of plant pigments. The term Chromatography can be translated literally from the Greek words chroma and graphein which means to ‘write with colours’. The technique remained peripheral to mainstream analytical techniques until the 1930s, when paper and thin-layer Chromatography (TLC) became popular. In the following decades, many novel chromatographic tech- niques were developed, making Chromatography the most widely used analytical technique in the biosciences. The diversity of chromatographic methods means that Chromatography can be used to purify any soluble (or volatile) compound if the correct adsorbent material, mobile phase and operating methods are employed. Chromatography can separate complex mixtures with impressive resolution; components such as proteins that may vary by only a single amino acid can be separated by Chromatography. These components are also immediately available for identification and quantification. If an important component has been identified, the whole chromatographic procedure can be ‘scaled up’ from a small-scale analytical run to a preparative run. Also, the conditions employed for Chromatography are typically not severe (gas/liquid Chromatography (GLC) is an exception), which allows for the analysis of sensitive components. 7.2 The Theory of Chromatography All compounds have different structures, and the properties of these different structures will influence the interaction a compound has with its immediate environment.
eBook - PDFThe Characterization of Chemical Purity
Organic Compounds
- L. A. K. Staveley(Author)
- 2016(Publication Date)
- Butterworth-Heinemann(Publisher)
Chromatography Space permits only an outline of the theoretical and practical details of Chromatography directed towards the particular interest of this book; any-one intending to use the technique should first study one of the numerous textbooks available 1 . Regular reviews are published in Analytical Chemistry 2 , and the gas chromatographer is especially well served for the study of original papers by Gas Chromatography Abstracts*. A collection of gas-chromatogra-phic data has been published by the A.S.T.MA The theory of chromato-graphy is treated in detail by Giddings 5 . The Chromatographie process may be described by the phases involved, and we have gas-liquid, gas-solid, liquid-liquid and liquid-solid chromato-graphy where the first word in each pair refers to the mobile phase. When a solid stationary phase is used, the separation process depends upon adsorp-tion; historically this was the first form of Chromatography but, particularly in gas Chromatography, liquid stationary phases have been more generally used and, for convenience in the discussion which follows, the liquid process, in which separation depends upon solubihty, will be more frequently referred to. The classification just given is not complete since Chromatographie separations can also be based on ion-exchange, electrophoresis and gel permeation, but they are not widely applicable to the substances with which this book is concerned. In gas Chromatography the moving phase is a gas, and is referred to as the carrier gas in conformity with the fact that (for practical purposes) there is no interaction between the molecules of the gas and those of the sample; this is not true in liquid Chromatography where the properties of the mobile phase (eluent, developer) are one of the factors determining the equilibrium between the phases.
eBook - PDF- James W. Robinson, Eileen Skelly Frame, George M. Frame II(Authors)
- 2014(Publication Date)
- CRC Press(Publisher)
845 CHAPTER 11 Principles of Chromatography 11.1 INTRODUCTION TO Chromatography Many.of.the.techniques.of.spectroscopy.we.have.discussed.in.the.preceding.chapters.are.. selective. for.certain.atoms.or.functional.groups.and.structural.elements.of.molecules . .Often,.this.selectivity.is. insufficient.to.distinguish.compounds.of.closely.related.structure.from.each.other . .It.may.be.insuf-ficient.for.measurement.of.low.levels.of.a.compound.in.a.mixture.of.others.that.produce.an.inter-fering.spectral.signal . .Conversely,.when.we.wish.to.measure.the.presence.and.amounts.of.a.large. number.of.different.compounds.in.a.mixture,.even.the.availability.of.unique.spectral.signatures. for.each.analyte.may.require.laborious.repeated.measurements.with.different.spectral.techniques. to.characterize.the.mixture.fully . .Living.systems.have.evolved.large.protein.molecules.(e .g., .recep-tors,.enzymes,.immune.system.antibodies).with.unique.3D.structures.that.can.bind.strongly.only.to. very.specific.organic.compounds . .Analytical.procedures.such.as.immunoassay,.enzyme-mediated. assays,.and.competitive.binding.assays.employ.the.extreme.selectivity.of.such.protein.macromol-ecules.to.measure.particular.biomolecules.in.very.complex.mixtures . .Microarrays.of.thousands.of. these,.each.with.unique.selectivity,.can.rapidly.screen.complex.mixtures.for.a.list.of.expected.com-ponents. .However,.precise.quantitation.of.each.detected.component.is.difficult.to.achieve.this.way . Analysis.of.complex.mixtures.often.requires.separation.and.isolation.of.components.or.classes. of.components . .Examples.in.noninstrumental.analysis.include.extraction,.precipitation,.and.distil-lation. . These. procedures. partition. components. between. two. phases. based. on. differences. in. the. components’.physical.properties . .In.liquid–liquid.extraction,.components.are.distributed.between. two.immiscible.liquids.based.on.their.similarity.in.polarity.to.the.two.liquids.(i .e., .“like.dissolves. like”).
- H. Ch. Curtius, Marc Roth(Authors)
- 2018(Publication Date)
- De Gruyter(Publisher)
114. E. Stahl (ed.). Springer-Verlag, Berlin, Heidelberg, New York (1969). 4 6 H. Determann 2. Column Chromatography H . DETERMANN a. Introduction Chromatography was first performed in columns (M. S. Tswett, 1903). Thin layer Chromatography, as described in the preceding chapter (II. A. 1.), can be looked at as a special case of column Chromatography. Thin layer plates are, in a sense, open columns with several advantages for analytical procedures. With respect to the column material, chromato-graphy in tubes is much more versatile, but some problems arise from the detection of separated fractions. In general, there are three properties of molecules that might be used for fractionation: solubility, size, and electrical charge. These properties generally overlap one another, since solubility depends in part on size and electrical charge. In clinical chemistry one normally deals with water soluble substances, so that the two parts of this chapter can be restricted to size and charge separation, respectively. These are special cases of the so-called liquid Chromatography (LC) which has found renewed interest since the pertinent equipment (columns, detectors and connective devices) has become available commercially. Their availability has led to an intense use of LC in all fields of analytical chemistry, including clinical chemistry. It is common practice in LC to use a vertical tube, equipped with some support for the filling material just above the bottom outlet and a solvent feeding system at the top. LC can be made very easy by selfassembling solvent reservoir, column, detector and/or fraction collector, or by combined instruments which contain all these items. The main difference between the two types is the speed of analysis. This is due to the high pressure which can be obtained in the integrated units by pumps and very tight connections.
eBook - PDF- Erich Heftmann(Author)
- 1991(Publication Date)
- Elsevier Science(Publisher)
In the process, the various sample components separate into zones (bands) that are touching. The final chromato- gram (No. 4 = displacer; Nos. 1-3 = sample components) is indicated in Fig. 1.28 by the arrow. Following the separation, a regenerant solvent is used to restore the column to its initial condition, allowing the entire process to be repeated. Displacement Chromatography has a number of potential advantages: larger samples and higher production rates, more concentrated fractions with less mobile phase to remove from purified products, etc. On the debit side, method development for this procedure is more complicated and difficult than in the case of elution (Figs. 1.25b and c). The relative importance of displacement vs. elution Chromatography for preparative sepa- ration is yet to be decided. (For a further account of displacement Chromatography, see Ref. 142.) REFERENCES 1 H. Purnell, Gas Chromatography,Wiley, New York, 1962. 2 J.C. Giddings, Dynamics of Chromatography, Part I, Principles and Theory, Dekker, New York, 1965. 3 L.R. Snyder, Principles of Adsorption Chromatography, Dekker, New York, 1968. 4 F. Helfferich and G. Klein, Multicomponent Chromatography, Dekker, New York, 5 B.L. Karger, L.R. Snyder and Cs. Horvath, An Introduction to Separation Science, 6 J.R. Conder and C.L. Young, Physicochemical Measurement by Gas 7 AS. Said, Theory and Mathematics of Chromatography, Huethig, Heidelberg, 1981. 8 S.T. Balke, Quantitative Column Liquid Chromatography, Elsevier, Amsterdam, 1984. 9 P.J. Schoenmakers, Optimization of Chromatographic Selectivity, Elsevier, Amsterdam, 1986. 10 R. Kaliszan, Quantitative Structure-Chromatographic Retention Relationships, Wiley- Interscience, New York, 1987. 11 E. Katz (Editor), Quantitative Analysis Using Chromatographic Techniques, Wiley, New York, 1987. 12 Cs. Horvath, in E. Heftmann (Editor), Chromatography, Part A, Elsevier, Amsterdam, 1983, Ch.
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